frequently asked questions

We are here to help you enjoy your scientific exploration. Please read these FAQs before contacting us. If your question still has not been answered, send us an email at support@thewellbio.com. For first time user of VitroGel system, please click to read the “first-time user note” to ensure optimal results.

GENERAL Questions

Which VitroGel® hydrogel product should I choose?

We currently have three hydrogel products: VitroGel 3D, VitroGel 3D-RGD and VitroGel RGD-PLUS.

All of our hydrogel products are ready-to-use and tunable.

  • VitroGel 3D is a pure and unmodified hydrogel which allows the maximum flexibility to manipulate the 3D cell culture environment for different needs. The unmodified hydrogel matrix structure is good for cell spheroid formation, suspension cells or cells require low cell-matrix interactions.
  • VitroGel 3D-RGD is modified with RGD cell adhesive peptide, promoting the cell attachment and cell-matrix interactions during the 3D cell culture. This RGD modified hydrogel system is good for adhesion cells or cells need good cell-matrix interactions.
  • VitroGel RGD-PLUS is  modified with a high concentration of RGD cell adhesive peptide, promoting the cell attachment and cell-matrix interactions during the 3D cell culture. This hydrogel system has 3X RGD peptide than VitroGel 3D-RGD, which maintains a high level of integrin binding activities even after hydrogel dilution.

We also have different functional ligand modified hydrogel (such as laminin or collagen) in our pipeline. Please contact at support@thewellbio.com if you need a customized product.

Can I harvest cells from the hydrogel after 3D culture?

Yes, cells can be harvested after 2D coating or 3D culture with the VitroGel™ Cell Recovery Solution. The solution is a ready-to-use, enzyme-free and can recover cells from VitroGel in 15 minutes. VitroGel Cell Recovery Solution is room temperature stable, has a neutral pH and works at 37 °C. The solution maintains high cell viability during the recovery process. After recovery, cells can be sub-culture in both 2D and 3D culture.

Please download the full user handbook and read the section on harvesting cells from hydrogel for details

Can I add extracellular matrix proteins or other molecular compounds into VitroGel?

Yes. Extracellular matrix proteins or other molecular compounds can be added into the VitroGel system. Before hydrogel formation, add the proteins or the molecular compounds into the cell culture media and then mix with VitroGel hydrogel solution. Please note that the hydrogel formation time and the final gel stiffness might change due to the salts contained in the proteins or chemical compounds. Please contact us at support@thewellbio.com if you have any questions or concern about adding additional compounds to the VitroGel system.

Can I do molecular analyses of protein and nucleic acids of cells cultured in VitroGel?

Yes. Cells can be harvested from the hydrogel and used to perform molecular analyses according to standard procedures. The hydrogel is transparent, you can use the molecular assay directly with hydrogel. Also, the hydrogel can be easily dissolve by using the ultrasonic processor, so scientist can lysis cells together with hydrogel to extract DNA/RNA.

Can cells grown in VitroGel be sub-cultured?

Yes. Cells can be harvested from the VitroGel system by using the VitroGel Cell Recovery Solution and sub-culture for an additional period by using fresh VitroGel.

How can I use VitroGel for co-culture or sandwich culture (layer by layer)?

Co-culture of two or more different cell types can be done with VitroGel system. Besides adding different cell types together with hydrogel solution for co-culture, each cell type can be also mixed with hydrogel solution and added layer by layer. After the first layer of hydrogel become stable, carefully overlay the second layer of cells/hydrogel mixture on top of the first layer of cells/hydrogel.

Is VitroGel compatible with staining and immunofluorescence protocols for confocal microscopy or other imaging technologies?

Yes. Cells can be stained within the hydrogel or harvested from the hydrogel and then stain. Most fluorescent dyes and immunological reagents can be used at standard protocols. Please read the full using handbook of VitroGel for more details. In addition, VitroGel hydrogel is transparent and compatible on different imaging systems for cell observation.

How long can cells grow in the VitroGel system?

We have tested the growth of cells in the hydrogel system for up to 6 weeks or more. Depended on the cell line, the 3D cell culture can last even longer. You might need to change the cover media more frequently once the number and size of cells increases.

Can I use VitroGel for in vivo studies?

Yes, VitroGel can be injected before or after hydrogel formation for in vivo study. Before hydrogel formation, the VitroGel solution can be injected directly into the animal which later becomes a hydrogel when it contacts with the ionic compounds of the physiological environment. Also, VitroGel hydrogel has an advanced injectable property after hydrogel formation. Mixing the hydrogel solution and cell culture media/PBS at a proper ratio (we recommend hydrogel solution: media/PBS = 3:1 or 4: 1 v/v), the final hydrogel becomes injectable for in vivo studies. Using this method, cells or other chemical compounds can mix well in the hydrogel before injection.

VitroGel Usage

How to prepare the cell suspension to mix the hydrogel? Shall I add serum?

If cells cultured in complete cell culture medium, which is supplement with 10% FBS or other critical supplement, please prepare the cell suspension using the following methods before mixing it with hydrogel solution.

  1. Prepare the cell suspension with 2X concentration (e.g. 100K), and mix with 100% FBS at 1:1 (v/v) ratio to get 1X cell suspension (50K) with 50% FBS.
  2. Mix the diluted hydrogel solution with the cell suspension from above at 4:1 (v/v) ratio to get the final cells in the hydrogel at 10K with 10% FBS supplement.

If serum plan is an important role in your traditional cell culture, it is also important for 2D coating and 3D culture. Adding serum supplement in the hydrogel and adjusting the final serum concentration to the target level would support cell growth in hydrogel system.

How do I adjust the stiffness of the final hydrogel?

The stiffness of the final hydrogel can be adjusted by diluting the hydrogel solution before mixing with cell culture media. Our VitroGel Dilution Solution can help to adjust the hydrogel concentration. Please read the “First-time User Note” to learn how to prepare different VitroGel dilutions. If you need a higher hydrogel stiffness than the original product, please contact us at support@thewellbio.com.

How do I adjust the hydrogel formation time?
  • If VitroGel needs to be diluted more than 1:3 ratio, a longer waiting time (20-30 min) may be needed for soft gel formation. Using a higher volume of cell culture medium for mixing would help to accelerate the process of hydrogel formation.
  • If the hydrogel solidifies too fast after mixing with culture medium (showing as small solid gel chunk), adjust the mixing ratio by using less cell culture medium. For example, if mixing 4 mL diluted hydrogel solution with 1 mL cell culture medium lead to the solid gel chuck (particles), then mixing 4 mL diluted hydrogel solution with 0.5-0.8 mL cell culture medium would help to solve the issue.
  • On the other hand, if the hydrogel formation is too slow, which may happen when using low hydrogel concentration at 1:3 or 1:4 dilution or using cell culture medium with very low ionic concentration, adjust the mixing ratio by using more cell culture medium. For example, if mixing 4 mL diluted hydrogel solution with 1 mL cell culture medium lead to a slow hydrogel formation, then mixing 4 mL diluted hydrogel solution with 1.5-4 mL cell culture medium would help to solve the issue.
What can I do to keep bubbles from forming when mixing the gel solution with the cell culture medium?

The bubble issue is related to the increased solution viscosity after mixing the gel solution with cell medium. Here are some suggestions that can help to reduce the formation of bubbles:
1) Warm up the hydrogel solution to 37° C to reduce the viscosity of the gel solution.
2) Gently mix the gel solution and cell medium and pipette slowly along the wall of the well plate without introducing bubbles.
3) Quickly spin the mixing tube to get rid of bubbles.

Can I form a dome shape culture? Why does the hydrogel stick loosely to tissue culture plate?
  1. Adding the hydrogel as a dome shape is not recommended.  The dome shape may not stick at the bottom the culture plate for long term culture.  The hydrogel should cover the whole bottom of the well plate.  We suggest tilting the well plate after adding the hydrogel to make sure the whole bottom of the well plate is covered by the gel. Using a 96 well plate can reduce the using volume of the hydrogel to 30-75 µL/well. Adding the cover medium to feed the cells has no issue with molecular penetration.
  2. Using non-treated tissue culture plate, which has a more hydrophobic surface which reduces the attachment of hydrogel/cells on the surface of well-plate. For better performance, we suggest using treated tissue culture plate with the VitroGel.
  3. Not enough waiting time before adding the additional medium on the top of the hydrogel. After transferring the hydrogel to the well plate, please wait 15-30 min for hydrogel stabilization before adding the top medium. Adding the medium before the hydrogel is stable would disrupt the structure of hydrogel. The low concentration of the hydrogel, the longer waiting time needed.
  4. After the initial soft hydrogel formation (step 5), it is important to make sure the hydrogel is stable and attached to the bottom of the well plate before adding the cover medium (if the hydrogel is not stable, it might detached from the bottom of the well after adding the cover medium). The gel might be soft, do not shake the plate or keep the plate vertically.
How fast shall I transfer the sample from mixing tube to the culture plate?

The VitroGel Dilution Solution will slowly initialize the hydrogel formation, therefore prepare FRESH diluted VitroGel. The hydrogel formation become much faster after mixing the diluted VitroGel solution with cell culture medium.  After mixing with cell culture medium, we recommend to immediately transfer the mixture to the tissue culture plate.

If you have multiple samples with different hydrogel conditions or cell types to prepare, transferring mixture of sample 1 to the tissue culture plate before mixing the hydrogel with cell culture medium for sample 2.

What seeding density should be used?

We recommend to use 200,000-1,000,000 cells/mL for this hydrogel system. The final cell density is highly depended on the research project. You might need to replace the covering culture media every 1-2 days accordingly.

How long before spheroid formation takes place?

Normally, spheroid formation requires about 5 – 10 days after culture in the hydrogel system. The formation time may vary depending on the cell line.